The present invention, in some embodiments thereof, relates to isolated PON1 polypeptides, polynucleotides encoding same and uses thereof in treating or preventing organophosphate exposure associated damage.
Inhibitors of acetylcholinesterase (AChE), including organophosphate (OP)-based pesticides and nerve agents, threaten both military and civilian populations. A timely pharmacological treatment with atropine and oxime AChE reactivators can save lives but in many cases does not prevent cholinergic crisis and the resulting onset of secondary toxic manifestations induced by OP intoxication. Side effects associated with drugs such as pyridostigmine used as protective treatment prior to OP exposure have also prompted the search for effective prophylactics and antidotes. Rather than minimizing the damages caused by the OP, the goal of prophylactic drugs is to intercept the OPs before they even reach their target organs. A stoicheiometric bioscavenger based on human butyrylcholinesterase has been recently developed. However, owing to the daunting mass ratio of OP to protein, hundreds of mgs of protein are required to confer protection against exposure to doses beyond a single LD50 dose [Ashani, Y. & Pistinner, S. Estimation of the upper limit of human butyrylcholinesterase dose required for protection against organophosphates toxicity: a mathematically based toxicokinetic model. Toxicol Sci 77, 358-67 (2004)]. Catalytic scavengers, namely enzymes displaying multiple turnovers, may allow rapid and efficient protection against high OP doses using low protein amounts [Ditargiani, R. C., Chandrasekaran, L., Belinskaya, T. & Saxena, A. In search of a catalytic bioscavenger for the prophylaxis of nerve agent toxicity. Chem Biol Interact [Epub ahead of print] (2010]. However, with few exceptions, xenobiotics such as OPs are promiscuous substrates for natural enzymes and are degraded with low catalytic efficiencies. Improved OP hydrolyzing enzyme variants have been engineered (e.g. PTE, DFPase, Hill, C. M., Li, W. S., Thoden, J. B., Holden, H. M. & Raushel, F. M. Enhanced degradation of chemical warfare agents through molecular engineering of the phosphotriesterase active site. J Am Chem Soc 125, 8990-1 (2003), Mee-Hie Cho, C., Mulchandani, A. & Chen, W. Functional analysis of organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides. Protein Eng Des Sel 19, 99-105 (2006), Melzer, M. et al. Reversed enantioselectivity of diisopropyl fluorophosphatase against organophosphorus nerve agents by rational design. J Am Chem Soc 131, 17226-32 (2009)), but prophylactic protection from ≧1XLD50 doses at reasonable protein amounts requires catalytic scavengers whose efficiencies in kcat/KM terms are ≧107 M−1 min−1.
The G-agents cyclosarin (GF) and soman (GD) comprise a prime target for, scavenger-based prophylaxis due to the low efficacy of pharmacological drugs used to counteract their toxicity [Kassa, J., Karasova, J. Z., Caisberger, F. & Bajgar, J. The influence of combinations of oximes on the reactivating and therapeutic efficacy of antidotal treatment of soman poisoning in rats and mice. Toxicol Mech Methods 19, 547-51 (2009)]. Although applied as racemates, their Sp isomers comprise the tangible threat (FIG. 5). Unfortunately, enzymes tested thus far primarily hydrolyze less toxic Rp isomer [Harvey, S. P. et al. Stereospecificity in the enzymatic hydrolysis of cyclosarin (GF). Enzyme and Microbial Technology 37, 547-555 (2005); Li, W. S., Lum, K. T., Chen-Goodspeed, M., Sogorb, M. A. & Raushel, F. M. Stereoselective detoxification of chiral sarin and soman analogues by phosphotriesterase. Bioorg Med Chem 9, 2083-91 (2001)].
Additional background art includes:
WO2004/078991
Alcolombri, U., Elias, M., and Tawfik, D. S. (2011). Directed evolution of sulfotransferases and paraoxonases by ancestral libraries. Journal of molecular biology 411, 837-853; Ashani, Y., Goldsmith, M., Leader, H., Silman, I., Sussman, J. L., and Tawfik, D. S. (2011). In vitro detoxification of cyclosarin in human blood pre-incubated ex vivo with recombinant serum paraoxonases. Toxicology letters 206, 24-28; and Gupta, R. D., Goldsmith, M., Ashani, Y., Simo, Y., Mullokandov, G., Bar, H., Ben-David, M., Leader, H., Margalit, R., Silman, I., et al. (2011). Directed evolution of hydrolases for prevention of G-type nerve agent intoxication. Nat Chem Biol 7, 120-125.